Comparative maternal protein profiling of mouse biparental and uniparental embryos

Abstract Background Maternal proteins have important roles during early embryonic development. However, our understanding of maternal proteins is still very limited. The integrated analysis of mouse uniparental (parthenogenetic) and biparental (fertilized) embryos at the protein level creates a protein expression landscape that can be used to explore preimplantation mouse development. Results Using label-free quantitative mass spectrometry (MS) analysis, we report on the maternal proteome of mouse parthenogenetic embryos at pronucleus, 2-cell, 4-cell, 8-cell, morula, and blastocyst stages and highlight dynamic changes in protein expression. In addition, comparison of proteomic profiles of parthenogenotes and fertilized embryos highlights the different fates of maternal proteins. Enrichment analysis uncovered a set of maternal proteins that are strongly correlated with the subcortical maternal complex, and we report that in parthenogenotes, some of these maternal proteins escape the fate of protein degradation. Moreover, we identified a new maternal factor-Fbxw24, and highlight its importance in early embryonic development. We report that Fbxw24 interacts with Ddb1-Cul4b and may regulate maternal protein degradation in mouse. Conclusions Our study provides an invaluable resource for mechanistic analysis of maternal proteins and highlights the role of the novel maternal factor Fbw24 in regulating maternal protein degradation during preimplantation embryo development.

activated using different methods, including high or low temperature, electrical or 89 chemical treatment [22]. The rates of oocyte activation depend on various factors, 90 including species, female age and culture conditions [22]. Also, methods of inducing  Among the quantified proteins, 1,298 proteins were detected in all six successive 134 developmental stages (Fig. 1B); however, few proteins were detected in only one 135 stage. For example, 57 proteins were only detected in the PA stage (after oocyte 136 activation) (Fig. 1B), including CCCTC binding factor, fcf1 rRNA processing protein,    Fig. 1E), including zinc finger protein 57 (Zfp57) and ring finger protein 2 (Rnf2). 163 The absence of Zfp57 in oocytes results in the failure of maternal methylation 164 imprinting at the Snrpn imprinted region; it is also required for the post-fertilization 165 maintenance of maternal and paternal methylation imprints at multiple imprinted 166 domains [33]. Rnf2 is a component of Polycomb-repressive complex 1 and functions 167 as a redundant transcriptional factor during oogenesis, which is essential for proper 168 zygotic genome activation (ZGA) [34].    Maternal proteins have different fates after oocyte activation, including degradation or 214 persistence. In this study, a comparison analysis was performed on the three proteome 215 databases that were constructed using mature oocytes (MII group), fertilized embryos 216 (biparental embryos, ZY group/ZY embryo) and parthenogenetic embryos 217 (uniparental embryos, PA group/PA embryo, without the paternal genome). In order to 218 eliminate the differences caused by different proteomic methods and to ensure the 219 reliability of these comparison results, we used the intersection of two reported MII 220 oocyte protein databases [16,18] Fig. 2A). The detailed 223 results of the Venn diagram in Fig. 2A are shown in Supplementary Table S4.  After artificial activation, 1,168 proteins stored in mature oocytes were also detected 241 in uniparental embryos (Fig. 3A). Correspondingly, 1,806 proteins stored in mature 242 oocytes were also detected in biparental embryos (Fig. 3B). Compared to the 243 biparental embryos, 613 proteins were only detected in uniparental embryos in this 244 study (Fig. 3C). Furthermore, to reveal the expression dynamics of these proteins 245 during development, Fuzzy c-means clustering was performed ( Fig. 3A   Intriguingly, a cluster of maternal proteins that was detected only in uniparental 256 embryos (PA group) in this study also followed this expression trend (cluster 1 in Fig.   257 3C), suggesting that embryonic compaction after the 8-cell stage is accompanied by 258 protein expression changes both in biparental and uniparental embryos. analyzing current 'omic' data, which can screen out potential key proteins or genes.

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The establishment of embryo proteomes during embryonic development provides data 285 support for such analyses. As mentioned before, five components of the SCMC (Ooep, Nlrp5, Tle6, Zbed3 and 288 Padi6, designated as SCMCs in this study) were detected and presented similar 289 patterns in both mouse biparental and uniparental embryos (Fig. 2B). In this study,  Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was 305 conducted on 429 candidate proteins (Fig. 4B). These proteins were found to be 306 widely involved in the regulation of many biological processes during embryonic information processing and cellular processes. The protein-protein interaction (PPI) 309 network of candidate proteins involved in 'Translation' was obtained using the 310 STRING database (https://string-db.org/), which shows a complicated interaction 311 protein network (Fig. 4C). To validate the expression trend of these candidate proteins, 312 eight candidate proteins were randomly selected (highlighted with a red box in Fig.   313 4C) for the PRM assay using biparental embryos. The results reveal that the protein 314 expression pattern coincided with the proteome results (Fig. 4D).  Table S7). These strongly negatively correlated proteins have complex interaction 320 networks ( Supplementary Fig. S3A). After GO annotation, it was found that these 321 proteins were mainly involved in translation initiation, mRNA transport and ribosome 322 biogenesis, and these proteins were mainly located in the nucleus. The molecular 323 functions of these proteins include translation initiation factor activity, protein  In this study, a group of candidate maternal proteins that strongly correlated with   Especially, 316 proteins were detected in MII oocytes and PA embryos, but not in ZY 380 embryos, indicating that these maternal proteins may be degraded after fertilization in 381 ZY embryos. However, in uniparental embryos, these maternal proteins seem to 382 escape degradation, which indicates that they may play a key role in parthenogenesis.

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In the normal bisexual reproduction of mammals, their function may only be in the    (Table 1). Obviously, the 421 quantity of Fbxws in biparental embryos is much more than in uniparental embryos.  (Table 1). Finally, Fbxw24 was selected for 429 further analysis, because its correlation relationship with the other Fbxws was similar 430 to that of SCMC components in biparental embryos (Table 1). This suggests that it 431 may be of importance in normal embryonic development from the zygote, and its role 432 in preimplantation embryo development has not been studied at all yet.  To study the underlying mechanism, we performed protein-protein interaction        To obtain fertilized embryos, super-ovulated female mice were mated with male mice.

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The mean ± standard error of the mean was used to express the data.

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The pronucleus stage was used as a control.  Table 1. The expression correlation coefficient of Fbxws and SCMC components.